Urinary tract infections (UTI) are quite common and may lead to permanent kidney damage if left untreated. The bacterium most often responsible for human UTI is Escherichia coli. E. coli isolates from symptomatic UTI often possess certain virulence-associated factors not as commonly found on E. coli's from human stool. These factors are adhesins (pili or fimbriae), the Col V plasmid, hemolysin and certain O and K polysaccharide antigens. Recently completed investigations by ourselves and our collaborators in Goteborg, Sweden, using isogenic mutants and transformants in a mouse ascending pyelonephritis model, have confirmed the importance of P-fimbriae and Type I pili in the colonization of kidneys and bladders, respectively. Although the addition of these adhesins to a fecal E. coli endowed it with a statistically significant increase in its ability to colonize the UT, the same additions to an E. coli K-l2 laboratory strain still did not permit it to colonize the mouse urinary tract. Using a combination of classical and recombinant genetic techniques, we propose to add the following factors, one at a time, to E. coli K-l2 P678-54: P-fimbriae and Type I pili, a UTI-associated O antigen, a UTI-associated K antigen, and the Col V plasmid. Isogenic sets of bacteria, differing in only one factor, will be inoculated in pairwise combinations into mouse bladders to compare the effectiveness of each factor in enhancing the ability of E. coli K-l2 to colonize the mouse urinary tract. Parallel genetic manipulations will be done to remove these factors from E. coli GR-l2, a human UTI strain with superior colonizing capacity compared to a fecal isolate harboring cloned adhesins (P-fimbriae and Type I pili). These experiments should identify which factor or factors are of paramount importance in the development of UTI, and may suggest potential methods for the prevention or treatment of UTI, such as identifying new targets for vaccine development or for intervention with specific chemical inhibitors.